Gene transfer method with the use of serum-free medium

ABSTRACT

A method for transferring a gene into target cells by a retrovirus with the use of serum-free medium. This method comprises infecting target cells with a retrovirus in serum-free medium optionally containing low-density lipoprotein and/or cytokines in the presence of a functional substance such as fibronectin in an amount effective in elevating the gene transfer efficiency of the retrovirus into the target cells by co-localizing the retrovirus and the target cells.

CROSS REFERENCE TO RELATED APPLICATION

This present application is the national stage under 35 U.S.C. 371 ofPCT/JP98/03173, filed Jul. 15, 1998.

TECHNICAL FIELD

The present invention relates to a gene transfer method with the use ofa serum-free medium, more specifically, to a method that elevates thegene transfer efficiency into target cells and enables efficienttransformation of the target cells, and to a series techniques concernedtherewith in the field of medicine, cell technology, gene technology,developmental technology and the like.

BACKGROUND ART

Mechanisms of a number of human diseases have been elucidated. Therecombinant DNA techniques and the techniques for transferring a geneinto cells have rapidly progressed. Under these circumstances, protocolsfor somatic gene therapies for treating severe genetic diseases havebeen recently developed. More recently, attempts have been made to applythe gene therapy not only to treatment of the genetic diseases but alsoto treatment of viral infections such as AIDS and cancers.

Most of the gene transfer trials in humans approved by Food and DrugAdministration (FDA) to date are ones in which a gene is transferredinto cells with the use of a recombinant retrovirus vector. Since theretrovirus vector efficiently transfers the foreign gene of interestinto cells and stably integrates the gene into their chromosomal DNAs,it is a preferable means of gene transfer particularly for the genetherapy in which a long-term gene expression is desired. This vector hasbeen subjected to various modifications so as not to have a badinfluence on the organism with the transferred gene.

For example, the replication function of the vector is deleted such thatthe vector used for the gene transfer does not replicate in the cellswhile repeating unlimited infection (gene transfer). Since such a vector(a replication-deficient retrovirus vector) cannot replicate by itself,a retrovirus in which the vector is encapsidated in a virus particle isgenerally prepared by using retrovirus producer cells (packaging cells).

Bone marrow cells are the preferable target cells for the somatic genetherapy since they can be handled in vitro and contain hematopoieticstem cells that are capable of self-replicating. It has beendemonstrated that umbilical cord blood also contains a number ofprogenitor cells including hematopoietic stem cells. By performing thegene therapy in which a gene is transferred into such target cells,which are then transplanted into an organism, the transferred gene canbe expressed in blood cells for a long period, and a disease can behealed for life.

However, the hematopoietic stem cell is one of the cells into which agene is not readily transferred with high efficiency, despite of thestudies by many groups. To date, the most efficient protocol for genetransfer with respect to hematopoietic stem cells from mice or otheranimals have been the method in which the hematopoietic stem cells areco-cultured with retrovirus producer cells. However, under theapprehension about safety, gene transfer in cell-free system with alower risk of contamination of the retrovirus producer cells has beendesired for clinical gene therapy methods for humans. Unfortunately, itis not easy to efficiently transfer a gene into hematopoietic stem cellswithout co-culturing with retrovirus producer cells.

Recently, it was reported that fibronectin, which is a component of theextracellular matrix, or a fragment thereof alone elevates the genetransfer efficiency into cells by a retrovirus (J. Clin. Invest.,93:1451-1457 (1994); Blood, 88:855-862 (1996)). Also, it has beendemonstrated that a fibronectin fragment produced by genetic engineeringtechnique has similar properties and can be utilized to efficientlytransfer a foreign gene into hematopoietic stem cells (WO 95/26200).

Furthermore, it is disclosed in WO 97/18318 that a functional substanceother than fibronectin (such as fibroblast growth factor, collagen etc.)elevates the gene transfer efficiency and that similar increase in thegene transfer efficiency is observed when a mixture of a functionalsubstance having a retrovirus-binding activity and another functionalsubstance having target cell-binding activity is used.

It is believed that the increase in gene transfer efficiency caused bythese functional substances is due to the increase in chance ofinteraction between the retrovirus and the target cells which areco-localized with the aid of the substances.

OBJECTS OF THE INVENTION

In the gene transfer methods using the retrovirus as described above,infection with the retrovirus (i.e., gene transfer) occurs when thetarget cells are cultured in a medium containing the retrovirus. Amedium containing a serum from an animal (in many cases, fetal calfserum (FCS)) is used in this step. Since the serum contains constituentsthat can serve as nutrients for cells and various growth factors, it isbelieved that the serum is highly effective for maintaining cells invitro.

It is believed that the component or the contents of constituents in theserum derived from an animal vary depending on the condition of thehealth or the like of the animal individual from which the serum wascollected. Therefore, reproducible results are not always obtained ifthe cultivation of the cells and/or the gene transfer is carried outusing sera of different lots. Furthermore, since a serum from aheterologous organism other than, for example, a human containssubstances antigenic against a human, suitable washing steps arerequired to decrease the contents of the antigenic substances when thecells maintained in the presence of such a serum are transplanted into ahuman. In addition, the quality of the serum must be strictly controlledsuch that the serum does not contain viruses, mycoplasmas or the like.

As described above, the gene transfer methods with the use ofserum-containing media have problems, of which the solution is desired.

SUMMARY OF THE INVENTION

The present inventors have studied intensively and surprisingly foundthat the use of serum-free medium elevates the gene transfer efficiencyas compared with a conventional serum-containing medium and that aparticularly high gene transfer efficiency is accomplished using amedium to which low-density lipoprotein is added. Thus, the presentinvention has been completed.

The first invention of the present invention relates to a method fortransferring a gene into target cells by a retrovirus, comprisinginfecting target cells with a retrovirus in serum-free culture medium inthe presence of a functional substance in an amount effective inelevating the gene transfer efficiency of the retrovirus into the targetcells by co-localizing the retrovirus and the target cells.

There is no limitation regarding the functional substance to be used inthe present invention. For example, a substance that has aretrovirus-binding site and a target cell-binding site in a singlemolecule or a mixture of a molecule that has a retrovirus-binding siteand another molecule that has a target cell-binding site can be used.For example, a functional substance such as fibronectin, fibroblastgrowth factor, collagen and polylysin, or a substance having aretrovirus-binding activity equivalent thereto (e.g., a heparin-bindingsubstance other than those described above) can be used as thefunctional substance that has the retrovirus-binding site. As thefunctional substance that has the target cell-binding site, for example,a substance having a ligand that binds to the target cell can be used.

The functional substance preferable to the present invention includes,for example, a fibronectin fragment having a retrovirus-binding sitesuch as a heparin-II-binding region and a cell-binding site such as abinding region to VLA-5 and/or VLA-4. For example, the polypeptide ofwhich the amino acid sequence is shown in the SEQ ID NO:1 of theSequence Listing (CH-296) is a fibronectin fragment that has aheparin-II-binding region and binding regions to VLA-5 and VLA-4.

There is no limitation regarding a culture medium to be used in themethod of the present invention as long as it does not contain a serum.A medium prepared by mixing constituents required for the maintenanceand the growth of the cells except the serum can be used. For example, acommercially available serum-free medium may be used. The medium maycontain suitable proteins or cytokines. In particular, a mediumcontaining low-density lipoprotein (LDL) is preferable to the presentinvention.

The second invention of the present invention relates to a cell with atransferred gene, characterized in that the gene is transferred by themethod of the first invention. There is no limitation regarding the cellinto which the gene is transferred. Various available cells can be usedas the target for the gene transfer.

The third invention of the present invention relates to a method oftransplanting a cell with a transferred gene into a vertebrate,characterized in that the cell with the transferred gene of the secondinvention is transplanted into the vertebrate.

The fourth invention of the present invention relates to a culturemedium used for transferring a gene, characterized in that the culturemedium does not contain a serum and contains a functional substance inan amount effective in elevating the gene transfer efficiency of theretrovirus into the target cells by co-localizing the retrovirus and thetarget cells.

As the functional substance used for the fourth invention, those asdescribed above can be used. Preferably, for example, the polypeptide ofwhich the amino acid sequence is shown in the SEQ ID NO:1 of theSequence Listing can be used.

There is no limitation regarding the medium of the fourth invention aslong as it does not contain a serum. For example, a commerciallyavailable cell culture medium without a serum, preferably, a medium withlow-density lipoprotein added can be used. In addition, the medium mayoptionally contain suitable cytokines.

DETAILED DESCRIPTION OF THE INVENTION

A recombinant retrovirus vector is usually used in the method fortransferring a gene of the present invention. In particular, areplication-deficient recombinant retrovirus vector is preferably used.Such a vector is deficient in replication such that it cannotself-replicate in infected cells and is non-pathogenic. A retrovirusinto which the vector is packaged can invade into a host cell such as avertebrate cell (particularly, a mammalian cell) and stably integrates aforeign gene inserted within the vector into the chromosomal DNA.

In the present invention, the foreign gene to be transferred into thecells can be used by inserting it into the retrovirus vector under thecontrol of an appropriate promoter, for example, the LTR promoter or aforeign promoter present in the retrovirus vector. In addition, anotherregulatory element, such as an enhancer sequence, which cooperates withthe promoter and a transcriptional start site, may be present in thevector in order to accomplish the transcription of the foreign gene.Preferably, the transferred gene can additionally accompany a terminatorsequence placed downstream the gene.

The foreign gene to be transferred may be a naturally occurring gene oran artificially prepared gene. Alternatively, the foreign gene may beone in which DNA molecules of different origins are joined together byligation or other means known in the art.

Any gene of which the transfer into cells is desired can be selected asthe foreign gene to be inserted into the retrovirus vector. For example,a gene encoding an enzyme or a protein associated with the disease to betreated, an antisense nucleic acid or a ribozyme or a false primer (see,for example, WO 90/13641), an intracellular antibody (see, for example,WO 94/02610), a growth factor or the like can be used as the foreigngene.

The retrovirus vector used in the present invention may have a suitablemarker gene that enables the selection of cells with the transferredgene. For example, a drug resistance gene that confers resistance toantibiotics on cells or a reporter gene that makes it possible todistinguish cells with the transferred gene by detecting the enzymaticactivity can be utilized as the marker gene.

The vector that can be used includes, for example, known retrovirusvectors such as MFG vector (ATCC No. 68754), α-SGC (ATCC No. 68755) andthe like. MFG-nlsLacZ vector (Human Gene Therapy, 5:1325-1333 (1994)),which is used in Examples described below, contains β-galactosidase geneas a marker gene. Thus, cells into which a gene is transferred by meansof this vector can be confirmed by examining the enzymatic activity ofthe gene product using a suitable substrate such as5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal).

These vectors can be prepared as virus particles into which the vectorsare packaged by using a known packaging cell line such as PG13 (ATCCCRL-10686), PG13/LNc8 (ATCC CRL-10685), PA317 (ATCC CRL-9078), GP+E-86(ATCC CRL9642) and GP+envAm-12 (ATCC CRL9641) (U.S. Pat. No. 5,278,056),Psi-Crip (Proc. Natl. Acad. Sci. USA, 85:6460-6464 (1988)) and the like.

A serum-free medium is used in the method of the present invention. Asused herein, a serum-free medium means a medium for culturing animalcells which does not contain a serum derived from an animal including ahuman and is composed of substances of which the constituents arechemically defined. A medium to which a purified and identifiedsubstance that constitutes a serum is added is included in theserum-free medium according to the present invention. The basicconstituents of such a medium include amino acids, saccharides, energysources such as organic acids, vitamins, buffering constituents foradjusting pH, inorganic salts and the like. The medium may contain a pHindicator such as phenol red. A known medium such as Dulbecco's ModifiedEagle's Medium (DMEM), Iscoves Modified Dulbecco's Medium (IMDM) and thelike can be used as the basic medium, which are commercially available,for example, from Gibco-BRL.

Various constituents can be added to these media depending on the typeof the target cells for the gene transfer or other objects. For example,various cytokines can be added to the media in order to promote orsuppress the growth or the differentiation of the target cells. Thecytokine includes interleukines (IL-3, IL-6 etc.), colony-stimulationfactors (G-CSF, GM-CSF etc.), stem cell factor (SCF), erythropoietin,various cell growth factors and the like. Many of these cytokinesderived from humans are commercially available. Upon using thesecytokines, one having the activity of interest is selected, andoptionally used in combination with another.

Furthermore, the present inventors have demonstrated that particularlyexcellent gene transfer efficiency is accomplished when low-densitylipoprotein (LDL) is added to the medium. Low-density lipoproteinderived from human is commercially available, for example, from Sigma.As shown in Examples described below, when a gene transfer into cells bymeans of a supernatant containing a retrovirus is performed in aserum-free medium containing low-density lipoprotein, a higher genetransfer efficiency is accomplished as compared with that accomplishedwith a conventional serum-containing medium. In addition, gene transfercan be performed with still higher efficiency in the presence of thefunctional substance as described below.

The present invention is characterized in that target cells are infectedwith a retrovirus in the presence of a functional substance which canelevate the gene transfer efficiency of the retrovirus into the targetcells by co-localizing the retrovirus and the target cells.

Cells with the transferred gene can be obtained with high efficiencywhen the cells are infected with the retrovirus in the presence of aneffective amount of the functional substance. For example, thefunctional substances as described in WO 95/26200 and WO 97/18318 can beused as the functional substance.

As used herein, an effective amount is an amount effective to result intransformation of target cells through the gene transfer into the targetcells by a retrovirus. A suitable amount is selected depending on thefunctional substance to be used and the type of the target cells. Theamount can be determined, for example, by measuring the gene transferefficiency by the method as described herein. In addition, gene transferefficiency means the efficiency of transformation.

There is no limitation regarding the functional substance having theretrovirus-binding site to be used in the present invention. Forexample, heparin-II-binding region of fibronectin, fibroblast growthfactor, collagen, polylysin and the like, as well as a substancefunctionally equivalent to these functional substances such as afunctional substance having a heparin-binding site can be used.Furthermore, a mixture of the functional substances, a polypeptidecontaining the functional substance, a polymer of the functionalsubstance, a derivative of the functional substance or the like can beused.

There is no limitation regarding the functional substance having thetarget cell-binding site to be used in the present invention. Thefunctional substance includes a substance that has a ligand that bindsto the target cell. The ligand includes a cell adhesive protein, ahormone or a cytokine, an antibody against a cell surface antigen, apolysaccharide or a glycoprotein, a sugar chain in a glycolipid, ametabolite of the target cell and the like. Furthermore, a polypeptidecontaining the functional substance, a polymer of the functionalsubstance, a derivative of the functional substance, a functionalequivalent of the functional substance or the like can be used.

The functional substance as described above can be obtained fromnaturally occurring substances, prepared artificially (for example, byrecombinant DNA techniques or chemical synthesis techniques), orprepared by combining a naturally occurring substance and anartificially prepared substance. In addition, a mixture of a functionalsubstance that has a retrovirus-binding site and another functionalsubstance that has a target cell-binding site can be used for the genetransfer using the functional substances as described in WO 97/18318.Alternatively, a functional substance that has a retrovirus-binding siteand a target cell-binding site in a single molecule, which may beselected or prepared, can be used.

In the method of the present invention, for example, fibronectin, afibronectin fragment or a mixture thereof can be used. These functionalsubstances can be either naturally occurring or prepared by chemicalsynthesis. They can be prepared in a substantially pure form fromnaturally occurring substances by a method as described, for example, inJ. Biol. Chem., 256:7277 (1981), J. Cell Biol., 102:449 (1986) or J.Cell Biol., 105:489 (1987). In this regard, fibronectin or a fibronectinfragment as used herein means one that is substantially free of anotherprotein naturally associated with fibronectin.

Furthermore, useful information regarding the fibronectin fragment whichcan be used herein or the preparation thereof is described in J.Biochem., 110:284-291 (1991), which further reports the above-mentionedrecombinant fragment); EMBO J., 4:1755-1759 (1985), which reports thestructure of the human fibronectin gene; and Biochemistry, 25:4936-4941(1986), which reports the heparin-II-binding region of humanfibronectin.

The fibronectin or the fibronectin fragment as described herein can beprepared from recombinant cells as described generally, for example, inU.S. Pat. No. 5,198,423. Specifically, a fibronectin fragment containingthe heparin-II region, which is a retrovirus-binding site, such asCH-296 (the amino acid sequence is shown in the SEQ ID NO:1 of theSequence Listing) which is used in Examples described below andrecombinant polypeptides such as H-271, H-296, CH-271 and the like aswell as the method for obtaining them are described in detail in thespecification of the above-mentioned patent. These fragments can beobtained by culturing Escherichia coli strains deposited under accessionnumber FERM P-10721 (H-296) (the date of the original deposit: May 12,1989), FERM BP-2799 (CH-271) (the date of the original deposit: May 12,1989), FERM BP-2800 (CH-296) (the date of the original deposit: May 12,1989) and FERM BP-2264 (H-271) (the date of the original deposit: Jan.30, 1989) at the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, Ministry of InternationalTrade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,Japan, as described in the publication. In addition, fragments that canbe typically derived from these fragments can be prepared by modifyingthe plasmids harbored in these Escherichia coli strains by knownrecombinant DNA techniques. Among the fibronectin fragments describedabove, H-296 has a polypeptide of binding region to VLA-4, CH-271 has apeptide of binding region to VLA-5, and CH-296 has both of them (NatureMedicine, 2:876-882 (1996)).

As described in Examples below, the gene transfer efficiency in aserum-free medium is elevated in the presence of the fibronectin or thefibronectin fragment CH-296. Also, a similar gene transfer efficiencycan be accomplished by using, for example, a mixture of H-271, which isa fibronectin fragment having a retrovirus-binding region, and C-271,which is a fibronectin fragment having a cell-binding activity, asdescribed in WO 97/18318.

By infecting cells with a retrovirus in a serum-free medium in thepresence of an effective amount of the fibronectin, the fibronectinfragment or the mixture thereof, cells with transferred genes can beefficiently obtained. The fibronectin, the fibronectin fragment or themixture thereof may be immobilized on the surface of the culture vesselused for the infection with the retrovirus, for example. The infectionwith the retrovirus can be performed according to a conventional method,for example, by incubation at 37° C. in 5% CO₂. The conditions and theincubation time may suitably changed depending on the target cells orthe objects.

Target cells are not infected with a retrovirus when they are in G₀phase. Therefore, it is preferable to induce the cells into the cellcycle by pre-stimulating them. For this purpose, the target cells arecultured in the presence of a suitable target cell growth factor beforeinfecting the cells with the retrovirus. For example, a target cellgrowth factor such as interleukin(IL)-6, stem cell factor and the likeis used to pre-stimulate bone marrow cells or hematopoietic stem cellsfor gene transfer.

There is no limitation regarding the cell to be used as the target forthe gene transfer by the method of the present invention. For example,stem cells, hematopoietic cells, non-adhesive low-density mononuclearcells, adhesive cells, bone marrow cells, hematopoietic stem cells,peripheral blood stem cells, umbilical cord blood cells, fetalhematopoietic stem cells, embryogenic stem cells, embryonic cells,primordial germ cells, oocytes, oogonia, ova, spermatocytes, sperms,CD34+ cells, c-kit+ cells, pluripotent hematopoietic precursor cells,unipotent hematopoietic precursor cells, erythroid precursor cells,lymphoid mother cells, mature blood cells, lymphocytes, B cells, Tcells, fibroblasts, neuroblasts, neurocytes, endothelial cells, vascularendothelial cells, hepatocytes, myoblasts, skeletal muscle cells, smoothmuscle cells, cancer cells, myeloma cells, leukemia cells and the likecan be used. The method of the present invention is preferably utilizedfor hematopoietic cells that are available from blood and bone marrowbecause these cells are relatively easy to obtain and because thetechniques for culturing and maintaining them are established.

Particularly, if a long-term expression of the transferred gene isintended, blood precursor cells such as hematopoietic stem cells, CD34+cells, c-kit+ cells, pluripotent hematopoietic precursor cells and thelike are suitable as target cells.

By using the fibronectin or the fibronectin fragment (especially, thefibronectin fragment having the binding site to VLA-5 and/or VLA-4 as acell-binding site) as the functional substance, the gene can beefficiently transferred into cells expressing VLA-5 and VLA-4 on theircell surface such as hematopoietic stem cells and CD34+ cells.

As described above, the cells into which a gene is transferred by themethod of the present invention can be transplanted into an organism,thereby enabling a gene therapy in which a foreign gene is expressed invivo. Since the cells with the transferred gene obtained by the methodof the present invention do not contain proteins or impurities derivedfrom a serum from a heterologous animal, they are suitable fortransplantation into an organism. For example, a gene therapy usinghematopoietic stem cells as target cells can be carried out by thefollowing procedure.

First, a material containing hematopoietic stem cells, such as bonemarrow tissue, peripheral blood, umbilical cord blood and the like, iscollected from a donor. Although such a material can be directly used inthe gene transfer procedure, mononuclear cell fractions containinghematopoietic stem cells are usually prepared by means of densitygradient centrifugation and the like, or hematopoietic stem cells arefurther purified by utilizing cell surface marker molecules such as CD34and/or c-kit. The material containing the hematopoietic stem cells isinfected with a recombinant retrovirus vector, into which a gene ofinterest is inserted by the method of the preset invention, after beingpre-stimulated by using a suitable cell growth factor, if necessary. Thecells with the transferred gene thus obtained can be transplanted into arecipient, for example, by intravenous administration. Although therecipient is preferably the donor itself, allogenic transplantation canbe performed. For example, if the umbilical cord blood is used as thematerial, the allogenic transplantation is carried out.

Some of the gene therapies using hematopoietic stem cells as the targetcells are for complementing a deficient or abnormal gene in a patient,for example, the gene therapy for ADA deficiency or Gaucher's disease.In addition, a drug resistance gene may be transferred into thehematopoietic stem cells in order to alleviate the damage ofhematopoietic cells due to the chemotherapeutic agents used for thetreatment of cancer or leukemia, for example.

A tumor vaccination therapy, in which a gene for a cytokine istransferred into cancer cells, the ability of the cancer cells toproliferate are deprived, and the cells are then returned to the body ofthe patient to enhance the tumor immunity, is investigated as a genetherapy for cancer (Human Gene Therapy, 5:153-164 (1994)). In addition,attempts are made to treat AIDS by a gene therapy. In this case, aprocedure in which a gene encoding a nucleic acid molecule (e.g., anantisense nucleic acid or a ribozyme) which interferes with thereplication or the gene expression of HIV (human immunodeficiency virus)is transferred into T cells infected with HIV, which is the causal agentof AIDS, is considered (e.g., J. Virol., 69:4045-4052 (1995)).

As described in Examples below, cells with a transferred gene can beobtained with high efficiency by using the gene transfer method of thepresent invention. Since the preparation of the cells thus obtained donot contain a serum derived from a heterologous organism, the cells canbe transplanted into an organism without additionally performing aspecial procedure. Furthermore, since the component and the contents ofthe constituents in the medium do not vary as compared with those inmedia used in conventional methods, reproducible gene transfer can beaccomplished.

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

EXAMPLE 1 Preparation of CH-296

A polypeptide derived from human fibronectin, CH-296 (the amino acidsequence is shown in the SEQ ID NO:1 of the Sequence Listing), wasprepared from Escherichia coli HB101/pCH102 (FERM BP-2800) containingthe recombinant plasmid pCH102, which contains the DNA encoding thepolypeptide, according to the method as described in U.S. Pat. No.5,198,423.

EXAMPLE 2 Immobilization of Fibronectin and CH-296 onto a Plate

Fibronectin (Sigma) and CH-296 prepared as described in Example 1 weredissolved in phosphate buffered saline (PBS, BioWhittaker) to 50 μg/mland 100 μg/ml, respectively, then filtered through 0.22-μm filters(Minisarto filter 0.22 μm, Sartorius). 1 ml of CH-296 solution orfibronectin solution was added to a well of 12-well plate (Falcon). Theplate was then incubated at room temperature for 2 hours forimmobilization. The solution subjected to the immobilization wasexchanged for 2 ml per well of PBS containing 2% bovine serum albumin(BSA, Sigma). The plate was then incubated for further 30 minutes atroom temperature. After incubation, the plate was washed twice withHanks' buffered salt solution (Gibco-BRL) containing 25 mM HEPES(Gibco-BRL).

EXAMPLE 3 Preparation of Virus Supernatant

DMEM medium (BioWhittaker) containing 10% calf serum (JRH Bioscience)and 100 units/ml penicillin—100 μg/ml streptomycin (Gibco-BRL) was usedfor culturing A7.21 cells derived from Psi-Crip cells (Proc. Natl. Acad.Sci. USA, 85:6460-6464 (1988)), which produce the retrovirus vectorMFG-nlsLacZ (Human Gene Therapy, 5:1325-1333 (1994)). 3×10⁶ A7.21 cellswere inoculated into 100-mm dishes for tissue culture (Falcon) andcultured overnight. The medium was then removed and replaced by 5 ml ofRPMI medium (BioWhittaker) containing 10% fetal calf serum (FCS,BioWhittaker) or 5 ml of BIT-9500 medium (Stem Cell Technologies)containing 40 μg/ml of low-density lipoprotein (LDL, Sigma). Aftercultivation for 24 hours, the culture supernatants were collected andfiltered through 0.45-μm filters (Minisarto filter 0.45 μm, Sartorius).Interleukin-3 (IL-3, Amgen), interleukin-6 (IL-6, Amgen) and Stem CellFactor (SCF, Amgen) were added to the culture supernatants to 10 ng/ml,10 ng/ml and 100 ng/ml, respectively.

The titers of the virus solutions thus obtained were 1.7×10⁷ pfu/ml forRPMI medium containing 10% FCS and 1.8×10⁶ pfu/ml for BIT-9500 mediumcontaining 40 μg/ml of LDL as measured using Rat2 cells (ATCC CRL-1764)according to the method as previously reported (Cancer Gene Therapy,4:5-8 (1997)).

EXAMPLE 4 Isolation of CD34+ Cells

CD34+ cells were isolated from human peripheral blood mobilized bychemotherapeutics and G-CSF (Human Gene Therapy, 5:1325-1333 (1994)) byusing immunological magnetic separation method (Magnetic-Activated CellSorting, Miltenyi Biotec). The purity of the resulting CD34+ cells was95%.

EXAMPLE 5 Gene Transfer into CD34+ Cells

(1) Gene transfer by supernatant method

CD34+ cells were pre-stimulated prior to infection with a retrovirus.

Specifically, The CD34+ cells prepared in Example 4 were incubated inthe presence of 10 ng/ml of IL-3, 10 ng/ml of IL-6 and 100 ng/ml of SCFin either RPMI medium containing 10% FCS or BIT-9500 medium containing40 μg/ml of LDL for 48 hours. 3×10⁵ pre-stimulated CD34+ cells weresuspended in the virus solutions prepared in Example 3. The virussolutions were selected such that the medium used for preparing thevirus solution was the same as that used for pre-stimulating the CD34+cells. The cell suspensions were added to wells with nothing immobilized(untreated wells), wells with the immobilized fibronectin prepared inExample 2, and wells with the immobilized CH-296 in a 12-well plate. Thecells were cultured in the presence of 5% CO₂ at 37° C. for 2 hours.Polybrene (4 μg/ml, Sigma) were added to the untreated wells and thewells with the immobilized fibronectin. After 2 hours, new virussolutions (containing IL-3, IL-6 and SCF at the above-mentionedconcentrations) were added and the cells were cultured in the presenceof 5% CO₂ at 37° C. for further 22 hours. After cultivation, the cellswere collected by using a cell detachment buffer, suspended in RPMImedium containing 10% FCS or BIT-9500 medium containing 40 μg/ml of LDL,then cultured for 24 hours.

(2) Gene transfer by co-cultivation

On the day before the start of the co-cultivation, A7.21 cells weretreated with 10 μg/ml of ametycine (Choay-Sanofi) for 2 hours, thencollected using trypsin/EDTA (Gibco-BRL). The cell suspensioncorresponding to 2×10⁵ cells was added to a 12-well plate. CD34+ cellswere pre-stimulated in the presence of 10 ng/ml of IL-3, 10 ng/ml ofIL-6 and 100 ng/ml of SCF in either RPMI medium containing 10% FCS orBIT-9500 medium containing 40 μg/ml of LDL for 24 hours. 10⁵pre-stimulated CD34+ cells were added to the wells to which the A7.21cells had been added, and cultured in the presence of 10 ng/ml of IL-3,10 ng/ml of IL-6, 100 ng/ml of SCF and 4 μg/ml of polybrene in themedium used for the pre-stimulation (1 ml/well). After 72 hours,non-adhesive cells were collected and suspended in IMDM medium(BioWhittaker).

EXAMPLE 6 Assessment of Gene Transfer

250 cells with the transferred gene obtained in Example 5 were added perplate containing 0.5 ml of semisolid medium (Methocult H4230, Stem CellTechnologies) containing 0.9% methyl cellulose, 30% FCS, 1% BSA, 0.1 mMmercaptoethanol and 2 mM glutamine (procedures were performed intriplicate). 2 units/ml of erythropoietin (Amgen), 10 ng/ml of GM-CSF(Amgen), 10 ng/ml of G-CSF (Amgen), 10 ng/ml of SCF, 10 ng/ml of IL-3and 10 ng/ml of IL-6 were added to the medium.

The gene transfer efficiency was calculated from the enzymatic activityof β-galactosidase expressed from the nlsLacZ gene. After cultivationfor 21 days, the plates were directly stained with X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside). The number of coloniesstained with blue color was counted to determine the ratio of thestained colonies against the total colonies.

The ratio of X-Gal positive colonies thus determined is shown in Table1.

TABLE 1 RPMI + FCS BIT + LDL Untreated well  4.8% 15.5%Fibronectin-immobilized well 14.1% 24.6% CH-296-immobilized well 11.8%33.6% Co-cultivation method 29.6% 22.3%

The results described above demonstrate that the combination of thefibronectin or the fibronectin fragment CH-296 and BIT-9500 mediumcontaining LDL (i.e., a serum-free medium) enables the gene transferwith high efficiency.

1 1 574 PRT Homo sapiens 1 Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly ProAsp Thr Met Arg Val 1 5 10 15 Thr Trp Ala Pro Pro Pro Ser Ile Asp LeuThr Asn Phe Leu Val Arg 20 25 30 Tyr Ser Pro Val Lys Asn Glu Glu Asp ValAla Glu Leu Ser Ile Ser 35 40 45 Pro Ser Asp Asn Ala Val Val Leu Thr AsnLeu Leu Pro Gly Thr Glu 50 55 60 Tyr Val Val Ser Val Ser Ser Val Tyr GluGln His Glu Ser Thr Pro 65 70 75 80 Leu Arg Gly Arg Gln Lys Thr Gly LeuAsp Ser Pro Thr Gly Ile Asp 85 90 95 Phe Ser Asp Ile Thr Ala Asn Ser PheThr Val His Trp Ile Ala Pro 100 105 110 Arg Ala Thr Ile Thr Gly Tyr ArgIle Arg His His Pro Glu His Phe 115 120 125 Ser Gly Arg Pro Arg Glu AspArg Val Pro His Ser Arg Asn Ser Ile 130 135 140 Thr Leu Thr Asn Leu ThrPro Gly Thr Glu Tyr Val Val Ser Ile Val 145 150 155 160 Ala Leu Asn GlyArg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser 165 170 175 Thr Val SerAsp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro 180 185 190 Thr SerLeu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr 195 200 205 TyrArg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu 210 215 220Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys 225 230235 240 Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly245 250 255 Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg ThrGlu 260 265 270 Ile Asp Lys Pro Ser Met Ala Ile Pro Ala Pro Thr Asp LeuLys Phe 275 280 285 Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp ThrPro Pro Asn 290 295 300 Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr ProLys Glu Lys Thr 305 310 315 320 Gly Pro Met Lys Glu Ile Asn Leu Ala ProAsp Ser Ser Ser Val Val 325 330 335 Val Ser Gly Leu Met Val Ala Thr LysTyr Glu Val Ser Val Tyr Ala 340 345 350 Leu Lys Asp Thr Leu Thr Ser ArgPro Ala Gln Gly Val Val Thr Thr 355 360 365 Leu Glu Asn Val Ser Pro ProArg Arg Ala Arg Val Thr Asp Ala Thr 370 375 380 Glu Thr Thr Ile Thr IleSer Trp Arg Thr Lys Thr Glu Thr Ile Thr 385 390 395 400 Gly Phe Gln ValAsp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln 405 410 415 Arg Thr IleLys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln 420 425 430 Pro GlyThr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala 435 440 445 ArgSer Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro 450 455 460Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser 465 470475 480 Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu485 490 495 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg ProGly 500 505 510 Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr GluTyr Thr 515 520 525 Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser GluPro Leu Ile 530 535 540 Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu ValThr Leu Pro His 545 550 555 560 Pro Asn Leu His Gly Pro Glu Ile Leu AspVal Pro Ser Thr 565 570

What is claimed is:
 1. A method for transferring a gene into targethematopoietic stem cells by a retrovirus, comprising infecting targethematopoietic stem cells with a retrovirus in serum-free culture mediumin the presence of low density lipoprotein and a functional substancewhich has a retrovirus-binding site and a target cell-binding site in anamount effective in elevating the gene transfer efficiency of theretrovirus into target hematopoietic stem cells by co-localizing theretrovirus and hematopoietic stem cells, wherein the functionalsubstance is fibronectin, a fragment of fibronectin, or a mixture offibronectin fragments.
 2. The method according to claim 1, wherein thefunctional substance is a fragment of fibronectin having the amino acidsequence as shown in the SEQ ID NO:1 of the Sequence Listing.
 3. Themethod according to claim 1, wherein the functional substance isimmobilized onto a culture vessel.
 4. The method according to claim 1,wherein the culture medium contains a cytokine.
 5. The method accordingto claim 4, wherein the culture medium contains a cytokine selected fromthe group consisting of IL-3, IL-6 and SCF.
 6. The method according toclaim 1, wherein the hematopoietic stem cells are CD34+ cells.
 7. Themethod according to claim 1, wherein the retrovirus is a recombinantretrovirus containing a foreign gene.
 8. The method according to claim7, wherein the retrovirus is a replication-deficient recombinantretrovirus.